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Occurrence, Public Health Implications and
Detection of Antibacterial Drug Residues in
Cow Milk Amol R. Padol
1*, C. D. Malapure
2, Vijay D. Domple
3, Bhupesh P. Kamdi
4
1Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Bareilly (India)
2Division of Animal Nutrition, Indian Veterinary Research Institute, Bareilly (India)
3Division of Physiology and Climatology, Indian Veterinary Research Institute, Bareilly (India)
4Division of Pathology, Indian Veterinary Research Institute, Bareilly (India)
*E-mail: dramolpadol@gmail.com
Article history:
Received 26 February 2015
Received in revised form
05 March 2015
Accepted 20 March 2015
Abstract
In India, although antibiotics have been used in large quantities
for some decades, until recently the existence of these drugs in
the milk and meat has received a little concern. It is only in
recent years that a more extensive investigation of antibiotic
substances has been attempted in order to permit an assessment
of the public health risks pose by them. Antibiotic usage in
livestock production as therapeutics, prophylactics and as
growth promoter has become vital to the growing dairy
industry, since the prolonged or inappropriate usage of such
antibiotics may lead to residues appearing in milk, which pose
the risk of human health hazards and also interfere with the
processing of the milk and milk products. The administration of
antibiotics against bacterial infection is a significant driving
force for selection of resistant strains of bacteria, which can
spread from animal to human population and complicate the
therapeutic management of such infections. Despite the
numerous investigations performed, there is still a lack of
understanding and knowledge about antibiotic residues in the
milk and milk products. In this review we addressed the present
state of knowledge concerning the occurrence, fate, public
health hazards and the methods used to detect of antibiotic
residues in milk
Keywords:
Antibacterials residues
Detection methods, public
Health hazards
Introduction
The increasing human population and changing standard of living across the
globe demand for the more food and other resources. The practical solution for this was
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Padol et al., / Environ. We Int. J. Sci. Tech. 10 (2015) 7-28
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to adopt the practices to improve the agriculture production and industrial output. Several
measures have been initiated by the Indian government to increase the productivity of
livestock through various schemes and policies including Delhi Milk Scheme (DMS),
Key Village Scheme (KVS), Integrated Cattle Development Project (ICDP) and some
other programmes were launched to improve breeding, feed and fodder availability and
effective disease control which has resulted in increasing the milk production
significantly and at present, India stands first in the milk production (Kumar et al., 2011).
The average growth in the milk production is about 5 % per annum with sustained growth
in the availability of milk and milk products for the growing population. Dairy sector has
become an important secondary source of income for millions of rural families and has
assumed the most important role in providing employment and income generating
opportunities. Milk is an excellent source of nutrients and considered as a natures perfect
food (and as almost complete food) since the antiquity (Enb et al., 2009). Milk contains
the optimal balance of proteins, fats, carbohydrates, vitamins and minerals providing a
range of benefits for growth, immunity and development for the calves and also to
human.
Milk is, however a potential ready source for disease agents and other biological
and chemical contaminants. Animal products and byproducts can potentially be
contaminated with thousands of chemicals which are used in day today life for the
various purposes. The most promising residues in animal derived products include
pesticides, antibacterial drugs, hormonal growth promoters, industrial chemicals and
heavy metals (Unnikrishnan et al., 2005; Khaniki, 2007). Antibiotics are the most widely
used veterinary drugs for therapeutic and prophylactic purposes and also as growth
promoter in dairy animals which may appear in milk as residues for a certain time period
(Wassenaar, 2005). Food Safety and Standards Act, 2006 defines veterinary drug residues
as “the parent compounds or their metabolites or both in any edible portion of any
animal product and include residues of associated impurities of the veterinary drugs
concerned” (FSSA, 2006). The residues can also be defined as the substances having a
pharmacological action, their metabolites and other substances transmitted to animal
products that are likely to be harmful to human health (Serratosa et al., 2006). The
antibacterial residues are sometimes called as “bacterial inhibitory substances” because of
the microbiological basis of the screening tests frequently used to detect them and
specific bacterial organism inhibited by them (McEwen et al., 1992). The presence of
residues may be the result of failure to monitor the withdrawal periods, illegal or off-label
use of drugs and incorrect dosage levels or dosing schedule. Unauthorized antibiotic use
and lack of good veterinary practices may result in residues of these substances in milk
and tissues (Ivona and Mate, 2002; Paturkar et al., 2005; Gaare et al., 2012). The detailed
understandings behind occurrence of residue in milk are explained in the later in the text.
Safe levels of residues in milk and other animal products result from the
participation of all activities involved in the food chain „from stable to table‟ (Serratosa et
al., 2006). Milk producers, dairy cooperatives, personnel involved in processing,
marketing and the veterinarians are directly or indirectly responsible to provide the safe
milk for the society. Veterinary drugs are extensively used to promote the animal health,
control and treat the infection and to step up the production (Botsoglou and Fletouris,
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9
2001). Presence of any drug or antibiotic residue in milk and meat is considered as
illegitimate and also lead to economic losses to dairy industry. A consumer demands the
food which is free from chemical residues. Many countries have imposed regulatory
limits (maximum residue limit/MRL) on the level of various drugs used for treatment of
dairy animals. MRL could be defined as maximum concentration of residues in a product
(milk, meat, egg) considered by the regulatory authorities as without sanitary hazard for
the consumer and without effect on the manufacturing processes (Paturkar et al., 2005).
Antibacterials used in dairy animals
Several antibiotics have been isolated from various sources and being used to
control disease pathogens. These agents are not only used for treating the infectious
diseases, but also used as feed additives such as chemotherapeutic agents (antibiotics,
anthelmintics) and growth promoters (Serratosa et al., 2006; Babapour et al., 2012).
Antibiotics can be grouped by either their chemical structure or mechanism of action. The
most commonly used antibacterials in veterinary medicine include -lactams,
tetracyclines, aminoglycosides, macrolides, quinolones and sulfonamides (Mitchell et al.,
1998; Unnikrishnan et al., 2005). These antibacterials may be used singly or at times in
combination when treating dairy cattle. Antibacterials are administered by various routes
to the animals. The most common routes are parenteral, tropical, oral (sometimes through
water and feed), intra-mammary and intra-uterine. All of these routes may result to
residues in milk either parent drug or its metabolites (Hubbert et al., 1996). Among the
above mention antibacterials oxytetracycline, chloramphenicol and streptomycin are
commonly excreted through milk by virtue of their pharmacokinetic properties (Zahid
Hosen et al., 2010).
Mastitis is the most prevalent and economically important widespread disease in
cattle which requires intensive antimicrobial treatment (Mohsenzadeh and Bahrainpour,
2008). Treatment of mastitis with antibacterials is of greatest regulatory concern because
of possibilities of antibacterial residues in milk (Sandholm et al., 2009). Mastitis is
treated with antibiotics delivered directly into the udder and sometimes injecting those by
parenteral routes. Treated cows are required to be excluded from the milk supply for a
specific time period to ensure that antibiotic residues are not excreted in their milk.
Antibiotic residues enter the milk supply when treated cows are returned to the milking
herd before the withholding period or when a cow retains antibiotic residues in her
system for an extraordinary length of time. Therefore milk from treated cows must not be
marketed until the recommended withholding period has elapsed (Khaniki, 2007).
Observance with recommended withholding time helps minimizing the risk of antibiotic
residues to occur in milk which is the exclusive responsibility of dairy farmer.
Research has quite extensively examined that, the reasons for contamination of
bulk tank milk is bad management practices involving delivery of milk from treated cows
during the withholding period, insufficient information flow between milking personnel,
failures during milking, treatment failures or milking of dry cows after antibiotic
treatment at drying off (Doherr et al., 2007). Other major reasons for occurrence of drug
residues in milk are incorrect milking order of cows and insufficient cleaning of milking
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cluster or milking installation. Few cases of prolonged occurrences of residues in milk are
related to veterinary error and insufficient cleaning of milk contact surfaces after milking
of treated cows. The milking interval is reported to have more or less relation to residues
excretion in milk. Henschelchen and Walser (1983) reported significantly shorter
excretion periods for procain-penicillin G and oxytetracycline when cows were milked
with 2 hour intervals after treatment. Different milking intervals in cows milked with
automatic milking systems may influence the withdrawal period and excretion of
antibiotics in milk of treated cows (Knappstein et al., 2003).
In general, the health of cow and udder also has profound effect on excretion of
antibiotics in milk. The fibrosis of udder tissue in chronic mastitis leading to poor
distribution and absorption of penicillin cause higher concentrations and longer retention
of penicillin in milk of the affected quarters compared to healthy quarters (Edwards,
1964). Intravenous infusion of high dosages of ceftiofur in cows with experimentally
induced Escherichia coli mastitis resulted in significantly longer excretion of ceftiofur in
milk compared to healthy cows (Erskine et al., 1995). In a study Cagnardi and colleagues
(2010) evaluated the effect of udder health on persistence of cefoperazone in the systemic
circulation and mammary tissue after intra-mammary administration. They reported that,
in the cows with subclinical mastitis the retention time of cefoperazone is more than that
of healthy cow. The transfer of antibiotics from treated quarters to non-treated quarters
and subsequent excretion in milk has been described for a number of antibiotics (Aureli
et al., 1990). In India the off label use of antibiotics mainly dosages deviating from
recommendations of the drug manufacturer fall under the main reason for occurrence of
antibiotic residues in milk after the end of the withholding period in cows. Due to off-
label use of veterinary drugs or noncompliance withdrawal periods, much higher residue
levels might appear in the milk and milk products (Nisha, 2008) (The term off-label is
employed whenever a drug is used in a manner other that which it is licensed for). Off
label use might include giving the drug to a different species or by different route or
against the infection that it isn‟t licensed or recommended.
As prophylactics and growth promoters, the antibacterials are used at lower
concentrations than those used for treatment. Because prevention of disease transmission
and enhancement of growth and feed efficiency are critical in modern animal husbandry
practices, there has been widespread incorporation of antibiotics into animal feeds in
many countries including India (Khachatourians, 1998). The prolonged use at low
concentration encourages the production of antibiotic resistant strains of bacteria
(Simonsen et al., 1998; Lin et al., 2013). One example is the emergence of
fluoroquinolone resistant Campylobacter, one of several bacterial species that cause
severe food poisoning in humans (Cheng et al., 2012).
Beta-lactam antibiotics
It is interesting to note that the ß-lactam antibiotics, including the penicillins,
cephalosporins, carbapenems and others, make up the largest share of antibiotics used in
most countries (Kummerer, 2009). -lactam antibiotics are broad spectrum antibiotics
interfering with cell wall synthesis, used generally to treat Gram positive and Gram
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negative bacterial infections (Droumev, 1983; Sun et al., 2013). -lactam antibiotics
include penicillins, cephalosporins, carbapenems and monobactam group of antibiotics.
Among the beta-lactam antibiotics, penicillins and cephalosporins forms the major
category used in veterinary medicine and are frequently used for the treatment of animals
all over the globe.
Penicillins are the most commonly applied antibiotics for the treatment of bovine
mastitis (Haapapuro et al., 1997) which frequently results in their residues in milk. The
residues of these antibiotics in milk cause problems in dairy industries and human health
hazards (Ghidini et. al., 2002). Penicillins are not inactivated at pasteurization
temperature or on drying and may cause allergic reaction manifested by skin rashes in
very sensitive individuals at very low concentration of 0.03 IU/ml (Bjorland et al., 1998)
to 0.01 IU/ml (Waltner-Toews and McEwen, 1994) in milk. Cross reactivity is observed
between penicillins and cephalosporins for development of allergic reactions.
Approximately 4 % of patients with a history of penicillin allergy experience an
anaphylaxis reaction to a cephalosporin (Kelkar and Li, 2001) and patients with a history
of a penicillin related allergic event have a increased risk of a reaction when given a
sulfonamide or a cephalosporin (Apter et al., 2006). The carbapenems and aztreonam
shows merely cross reactivity with penicillin and other beta lactams (Romano et al.,
2007). Beta lactam antibiotics are sometimes associated with neurotoxicity manifested by
hallucinations, twitching, and seizures (Snavely and Hodges, 1984). Pre-existing brain
lesions, renal dysfunction and hyponatremia can trigger the neurotoxic symptoms even at
lower concentration of these antibiotics (Granowitz et al., 2008).
Tetracyclines
The tetracyclines are broad-spectrum antibacterials active against Mycoplasma,
Chlamydophila and Rickettsia in addition to bacteria. Tetracyclines are bacteriostatic and
acquired resistance is now widespread among bacteria (Fuoco, 2012). Tetracyclines may
be administrated parenterally, orally through feed or water or by intra-mammary infusion.
The widely used oxytetracycline and the less often used tetracycline and chlortetracycline
have similar properties. Fraction of tetracyclines execrated in bile gets reabsorbed
through entero-hepatic circulation, and may persist in the body for a long time after
administration (Chambers, 2006). The rate of metabolism of tetracyclines in cows has
been estimated to 25-75 % and a significant percentage of the administrated tetracyclines
are excreted in bovine milk (Abbasi et al., 2011). If these antibiotics administrate
improperly or if the withdrawal period for the treated cows has not been passed, the
parent drug and their metabolites may end up in milk and may cause harmful effects to
consumers (Fritz and Zuo, 2007). Demeclocycline, doxycycline and other tetracyclines to
a lesser extent associated with mild to severe photosensitivity reactions upon exposure to
sunlight in the individuals which are previously treated with these antibiotics. Photo-
onycholysis and pigmentation of the nails may develop with or without accompanying
photosensitivity (Chambers, 2006). Photo-onycholysis is a phototoxic reaction, which is a
drug-induced separation of the nail from the nail bed when exposed to ultraviolet
radiation. Tetracyclines can cross the placenta and enter the fetal circulation and amniotic
fluid. Relative to the maternal circulation, tetracycline concentrations in umbilical cord
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plasma and amniotic fluid are 60 % and 20 % respectively. Relatively high
concentrations of these drugs also are found in breast milk. Children receiving
tetracyclines for long or short duration may develop permanent brown discoloration of
the teeth (Navratilova et al., 2009). The larger the amount received relative to body
weight, the more intense the enamel discoloration. Exposure of pregnant women with
tetracyclines may produce discoloration of the teeth in her children.
Aminoglycosides
Aminoglycoside were the first antibiotics discovered by systematic screening of
natural product sources for antibacterial activity (Hermann, 2007). The laboratory of
Waksman reported the discovery and isolation of the streptomycin from soil bacteria
Streptomyces griseus in 1944, which was the first antibiotics used effectively against
Mycobacterium tuberculosis (Schatz and Waksman, 1944). Aminoglycosides are usually
used synergistically with β-lactams for the treatment of serious infections due to Gram
positive and Gram negative bacteria. They act by binding to the A site of the 30 S small
ribosomal subunit, inhibiting translation process in protein synthesis (Hermann, 2005).
The antibiotic, gentamicin in this class is used mainly to treat uterine infections in cattle,
particularly dairy animals. However, one of its side effects is its tendency to accumulate
and persist in bovine kidney tissue for several months (Elezov et al., 1984). Semi-
synthetic derivatives of natural products like amikacin, netilmicin and tobramycin have
been the most fruitful source of new clinically useful antibiotics (Von Nussbaum et al.,
2006). In humans, the nephrotoxicity of aminoglycosides is associated with small
portions of the drug being accumulated in the renal cortex and leading to reversible renal
impairment (Mingeot-Leclercq and Tulkens, 1999; Granowitz et al., 2008). Considering
the antibiotics induced nephrotoxicity, the aminoglycosides and amphotericin are the
prototypical classes associated with acute renal failure; however, other agents including
sulfonamides, beta-lactams and acyclovir, have been reported to cause renal impairment
in variable severity. In addition to nephrotoxicity, aminoglycosides can cause irreversible
ototoxicity that occurs both in a dose-dependent and idiosyncratic manner (Fischel-
Ghodsian, 2005). Some animal study data demonstrated the probable role of reactive
oxygen species to induce specific ototoxicity (Bates, 2003).
Sulphonamides
Sulphonamides are among the oldest groups of antibacterials widely used in the
treatment of bacterial and coccidial diseases of dairy cattle and as growth promoter in
swine. They have a wide spectrum of bacteriostatic action, effective against both Gram
positive and Gram negative organisms. These types of antibiotics dissolve quickly, easily
distribute in all tissues and body fluids, including the cerebrospinal fluid and fetal
circulation. About 90 % of these antibiotics bind to plasma proteins, and their maximum
concentration seen 3-6 hours after the adminstration. They are metabolized primarily in
the liver and excreted in the urine by glomerular filtration. The primary mechanism of
adverse effects produced by these antibacterials in humans is associated with the
impairment of thyroid-hypothalamus-pituitary axis and should be assessed by measuring
parameters of thyroid and pituitary function (JECFA, 1990). Sulfonamides are also
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known to produce sensitization and are one of the most common classes of antibacterial
that produce allergic reactions at therapeutic dose (Golembiewski, 2002), but there have
been no cases of human allergies that involved exposure to residues in animal foods
(Paige et al., 1999).
Chloramphenicol
Chloramphenicol is a broad spectrum antibiotic, isolated from bacterium
Streptomyces venezulae in the year 1947. It is clinically used to treat chronic infection of
respiratory tract, bacterial meningoencephalitis, brain abscesses and intraocular infections
and is active against a variety of pathogens including bacteria, Spirochaetes and
Rickettsiae (Shukla et al., 2011). Chloramphenicol and its metabolites may appear in
milk after parenteral administration, however after oral administration to cattle it is not
excreted in milk (De Corte-Baeten and Debackere, 1976). Chloramphenicol has been
associated in a non-dose related manner with aplastic anemia (Shukla et al., 2011) and
bone marrow suppression (Ambeker et al., 2000) in a small proportion of human to
whom the drug was exposed. Some of the individuals who survive the bone marrow
depression may develop leukaemia, which creates concerns about possible
carcinogenicity (Doody et al., 1996). Although, most countries have banned the use of
chloramphenicol in food and dairy animals, nevertheless traces of it have been detected in
shrimp and other aquaculture products (Shukla et al., 2011).
Other antibacterials
Nitrofurans are the broad spectrum synthetic antibacterials, primarily used to treat
Gram positive bacterial infections. In veterinary medicine these are used for topical
application on infected burns and skin infections e.g. nitrofurazone (Vasheghani et al.,
2008) for the oral treatment of cholera e.g. furazolidone (Roychowdhury et al., 2008),
bacterial diarrhoea and giardiasis (Petri, 2005); and to treat urinary tract infections e.g.
nitrofurantoin (Guay, 2008). These antibacterials are sometime associated with adverse
reactions involving hemorrhagic diathesis, anemia, nausea and vomition in livestock and
cardiotocicity in birds. Animal studies demonstrated the mutagenic (Ahmed et al., 2008)
and neurotoxic effects of long term exposure to nitrofurans. The possible mechanism
linked to the mutagenic effect of furazolidone and other nitrofurans involves the
irreversible damage to the DNA of epithelial cells as well as endocrine dysfunction
occurred prevalently when cells are exposed to furazolidone (De Angelis et al., 1999).
Based on animal study data, nitrofurans and some anti-parasitic drugs, such as
dimetridazole, also raise some concern of carcinogenicity (Waltner-Toews and McEwen,
1994).
Another synthetic class, fluoroquinolones are broad spectrum antibacterials
primarily active against Gram negative pathogens. These are effective for the therapy of
serious infections, e.g. septicemia, gastroenteritis and respiratory diseases and also used
for the treatment of infections of the urinary tract and soft tissues (Nizamlıoglu and
Aydın, 2012). They are effective in the therapy of mycoplasma infections and infections
caused by atypical bacteria (Navratilova et al., 2011). In veterinary medicine, they are
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14
useful especially in the therapy for gastrointestinal and respiratory tract infections
(Botsoglou and Fletouris, 2001), enrofloxacin being the most widely used
fluoroquinolone in veterinary medicine (Monica et al., 2011). Fluoroquinolone
preparations are also used for the prevention and treatment of mastitis in lactating cows
and for dry cow therapy (Gruet et al., 2001).
A few antibacterials are also known to cause functional changes in the rhythm and
rate of cardiac contraction. Macrolides, some quinolones, azoles, pentamidine and
quinine can prolong the Q-T interval of the cardiac cycle and cause ventricular
arrhythmia. Individual receiving oral erythromycin is found to have increased risk of
sudden death probably due to Q-T interval prolongation (Ray et al., 2004). On the other
hand erythromycin, azithromycin and other macrolides can cause dose dependant and
serum concentration dependent ototoxicity associated with bilateral hearing loss or
labyrinthine dysfunction (Swanson, 1992; Coulston and Balaratnam, 2005; Hajiioannou
et al., 2011) and sometimes may results in permanent hearing loss or vertigo (Ress and
Gross, 2000).
Public health hazards imposed by antibacterial residues
The prevalence of food borne illness is increasing worldwide and it has a major
public health impact (Nguz, 2007). Food borne illnesses are caused by eating or drinking
beverages and other food articles contaminated with bacteria, parasites, viruses or
chemical contaminants usually as a result of food mishandling and mismanagement
practices. In India insufficient data is available regarding the food borne hazards to
human and environment. In China the estimated incidents of food borne hazards was 200-
400 thousand people annually (Xie and Yongda, 2002). Centers for Disease Control and
Prevention (CDC) estimated that food borne illnesses affect 48 million people each year
in United States (CDC, 2011).
Most of the antibacterials currently used in the control and treatment of farm
animal diseases are relatively non toxic even at higher concentration, but there are few
antibiotics which pose a significant threat to public health when present in sufficiently
high concentrations in food (McEwen and McNab, 1997). Antibiotic residues in milk are
of great public health concern since milk is being widely consumed by infants, youngster
and adults throughout the globe (Khaniki, 2007). Considering the issue of public health
hazards, milk and milk products contaminated with antibiotics and other chemical
contaminants beyond a given residue levels, are considered unfit for human consumption
(Hillerton et al., 1999; Goffova et al., 2012). Occurrences of veterinary drug residues
pose the broad range of health consequences in the consumers. The residues of
antibacterials may present pharmacological, toxicological, microbiological and immune-
pathological health risks for humans (Drackova et al., 2009). In rare situations, the
pathogens against which the antibiotics are being used have less public health hazards
than those posed by the improper use of antibiotics. To enlist one example, mastitis
pathogens in milk pose a lower threat to public health if milk is pasteurized. On the other
hand, the careless antibiotic therapy to eliminate mastitis pathogens becomes a public
health concern due to their residues in milk [84] (Hameed, 2006).
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Antibacterial causes broad range of health effects, to summarize they can cause
development anomalies e.g. bone marrow aplasia (chloramphenicol) and can alter the
normal gastrointestinal microflora resulting in GI disturbances (intestinal dysbiosis) and
development of resistant strains of bacteria. Therefore, the use of antibacterials may
result in emergence of antibiotic resistant strains of pathogens, complicating the treatment
for both human and animal diseases (Dewdney et al., 1991; Goffova et al., 2012). In
addition some of the antibacterial may act as carcinogens and pro-carcinogens
(Oxytetracycline and furazolidone). Carcinogenic and genotoxic effects in consumers
could be possible at very low exposure levels, especially if the chemical in question is
ingested regularly over a long period of time. Oxytetracycline can react with nitrite and
the combination thereof called as nitrosamine is a potential carcinogen (Mitchell et al.,
1998). In the year 1995 European Union (EU) prohibited the use of nitrofurans for the
treatment of bacterial diseases in livestock production, due to concerns about the
carcinogenicity of their residues in edible tissue (Vass et al., 2008). In subsequent years
Australia, USA, Philippines, Thailand and Brazil also prohibited the use of nitrofurans in
food animals (Khong et al., 2004).
These hazards can be categorized in to two types as direct-short term hazards and
indirect-long term hazards, according to duration of exposure to residues and the time
onset of health effects (Muhammad et al., 2009). The direct health hazards includes the
health effects caused due to excretion of drug in milk, as an example the beta-lactam
group of antibiotics regardless of their low concentration in milk causes allergic
hypersensitive reaction in sensitized individual immediately after consumption (Paige et
al., 1997; Sierra et al., 2009). Another example includes the aplastic anemia caused by
chloramphenicol not related to level of exposure (Rich, 1950; Granowitz et al., 2008).
Several antibiotics are potent antigens or act as a haptens and occupational
exposure on a daily basis can lead to allergic reactions. Most of the reported allergic
reactions are related to ß-lactam antibiotic residues in milk or meat and the allergic
reaction has been associated with exposure to antibiotic residues in foods. Many of the
cases refer to people previously treated with antibiotics and hypersensitized to a degree
that subsequent oral exposure evoked a response. A hypersensitivity reaction to a drug is
either IgE-mediated or Non–IgE-mediated reactions. IgE-mediated reactions occur
shortly after drug exposure. Instances of IgE-mediated hypersensitivity reactions include
urticaria, anaphylaxis, bronchospasm and angioedema. Non IgE-mediated reactions
include hemolytic anemia, thrombocytopenia, acute interstitial nephritis, serum sickness,
vasculitis, erythema multiforme, Stevens-Johnson syndrome and toxic epidermal
necrolysis (Granowitz et al., 2008).
Indirect and long term hazards are the effects caused by long term exposure of an
individual to residues and include carcinogenicity, teratogenicity and reproductive
effects. Long term exposure to diethyl stilbesterol can cause vaginal clear cell
adenocarcinoma and benign structural abnormalities (Offman and Longacre, 2012). The
long term exposure to antibiotic residues in milk may result in alteration of the drug
resistance of intestinal microflora (Ram et al., 2000). The use of the antibiotic avoparcin
as a growth promoter in food animals resulted in the development and amplification of
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16
vancomycin resistant enterococci. Subsequent colonization in human intestine of these
resistant strains causes the clinical disease that would be difficult to treat (Tzavaras et al.,
2012). Even if the resistant strains bacteria are not human pathogens, they may still be
dangerous because they can transfer their antibiotic resistance genes to other pathogenic
bacteria (Hayek, 2013). Antibiotic resistant strains of bacteria, including Salmonella, E.
coli and Campylobacter spp., have been isolated from farm animals in many countries
(Aarestrup et al., 1997; Brichta-Harhay et al., 2011).
Antibacterial agents like tetracyclines, nitrofurans and sulfonamides are used as
feed additives in cattle feed which may excrete in milk and sometimes associated with
toxicological effects in human (DeVries, 1997). Carcinogenic and mutagenic effects were
demonstrated on animals due to nitrofurans used at high and lengthened doses (De
Angelis et al., 1999; Gutierrez et al., 2011). These antibacterials are now prohibited in
most countries for veterinary use in animal production. The other substances considered
as potentially carcinogenic are antibiotic additives: quinoxaline, carbadox and olaquindox
(FAO/WHO, 1995). In long term animal studies, amoxicillin (Abou-Tarboush, 1994),
chloramphenicol (Fritz and Hess, 1971), doxycycline (Schaefer et al., 1996), gentamicin
(Nishio et al., 1987) and rifampin (Holdiness, 1987), have shown effects on reproduction
performance in parents and developmental toxicity in their offsprings. This data hold
functional insight on possible adverse effects of these antibacterials in pregnant and
lactating women and to their young ones. Recently in a study, Etminan and coworkers
(2012) postulated the risk of retinal detachment in individuals upon continued exposure
to fluoroquinolones. Chloramphenicol is also associated with optic neuropathy (Wong et
al., 2013) and brain abscess (Wiest et al., 2012) with varied intensities and clinical
manifestations.
Apart from the health hazards, antimicrobial residues in milk are responsible for
interference with starter culture activity and hence disrupt the manufacture process of
milk products (Katla et al., 2001). In the fermented dairy products manufacturing plants,
such as cheeses and yogurts, the presence of antimicrobial agents can lead to the partial
or total inhibition of the lactic bacterial growth. Antibiotic residues can also interfere with
the methylene blue test, intended to estimate the total microbial load in milk. The time
taken for reduction of the dye will be increased, hence causing under estimation of the
microbial load. All of these concerns may result in major economic losses to the dairy
industry.
Antimicrobial residues and regulatory issues in India
Milk offered for sale is a specific food item that requires strict control and
enforcement by all legal regulations and relevant authorities that must allow its marketing
at home country and abroad only through special and official permission.
In regard to dairy animals in developed countries, to avoid risks related to drug
residues in milk, in many countries maximum residue limits (MRLs) have been
established for each antimicrobial agent by law, below which it is considered that the
drug may safely be used without harming the consumer. In the European Union, the
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Table.1. Maximum Residue Limits (MRL) for commonly used veterinary drugs in cow
milk (Codex Alimentarius Commission 2012)
Sr.
No. Veterinary drug Class of drug MRL (μg/l)
1 Albendazole Benzimidazole anthelmintic 100
2 Amoxicillin Penicillin antibiotic 4
3 Benzyl-penicillin Penicillin antibiotic 4
4 Ceftiofur Cephalosporin antibiotic 100
5 Chlortetracycline Tetracycline antibiotic 100
6 Oxytetracycline Tetracycline antibiotic 100
7 Tetracycline Tetracycline antibiotic 100
8 Colistin Polymyxin antibiotic 50
9 Dihydrostreptomycin Aminoglycoside antibiotic 200
10 Streptomycin Aminoglycoside antibiotic 200
11 Diminazene Aromatic diamidine trypanocide 150
12 Doramectin Avermectin anthelmintic agent 15
13 Eprinomectin Avermectin anthelmintic agent 20
14 Febantel Phenylguanidine anthelmintic agent 100
15 Fenbendazole Benzimidazole anthelmintic agent 100
16 Oxfendazole Benzimidazole anthelmintic agent 100
17 Gentamicin Aminoglycoside antibiotic 200
18 Imidocarb Carbanalide antiprotozoal agent 50
19 Isometamidium Trypanocide 100
20 Ivermectin Avermectin anthelmintic agent 10
21 Lincomycin Lincosamides antimicrobial 150
22 Monensin Polyether ionophores antimicrobial 2
23 Neomycin Aminoglycoside 1500
24 Pirlimycin Lincosamide antibiotic 100
25 Spectinomycin Aminocyclitol antibiotic 200
26 Spiramycin Macrolide antimicrobial 200
27 Sulfadimidine Sulfonamide antimicrobial 25
28 Thiabendazole Benzimidazole anthelmintic agent 100
29 Tylosin Macrolide antimicrobial 100
30 Procaine benzyl-penicillin Penicillin antibiotic 4
Padol et al., / Environ. We Int. J. Sci. Tech. 10 (2015) 7-28
18
MRLs in food of animal origin are established by the Codex Alimentarius Commission
(Codex Alimentarius Commission, 2012). These MRLs for commonly used veterinary
drugs as listed in the table 1 would be followed by the countries wherein the regulatory
limits are not established by the concern authorities. In India the manufacturing of
antibiotics and other drugs for human and veterinary use are regulated by Central Drug
Standard Control Organization (CDSCO) under Drugs and Cosmetics Act of 1940 and
the rules therein. However, there is intrinsically no regulation especially for use of
antibiotics in animals for treatment and as growth promoter. Multiple regulations for food
have been ordained at different points of time to supplement each other. This incremental
approach has lead to incoherence and inconsistency in the food sector regulatory
scenario.
The Food Safety and Standards Authority of India (FSSAI) under the Ministry of
Health and Family Welfare is the main authority for establishing the scientific standards
for articles of food including milk and milk products and to regulate their manufacture,
storage, distribution, sale and import, to ensure availability of safe and wholesome food
for human consumption and for matters connected therewith as per the rules specified by
Food Safety and Standard Act, 2006 (FSSA, 2006). The Food Safety and Standards Act,
2006 is a new legislative act which replaced the Milk and Milk Products Order, 1992 on
August 5, 2011. The FSSA integrates eight different existing food laws, and is a major
transformation that ensures to bring paradigm shift in the food regulatory scenario in
India. The FSSAI and State Food Authorities are responsible for implementation and
enforcement of the FSSA. The Section 21(1) of FSSA, 2006 specifies that the “food
should not contain any insecticides or pesticides residues, veterinary drugs residues,
antibiotic residues, solvent residues, pharmacological active substances and micro-
biological contaminants in excess of limits prescribed under the regulation” (FFSA,
2006). FSSAI have recognized few laboratories from different regions of country for
complete analysis of the food samples for presence of as per the Food Safety and
Standards (Food Products Standards and Food Additives-Part-I & II) Regulations, 2011
including the chemical contaminants, mycotoxins and veterinary drug residues.
Methods for detection of antibacterial residues in milk
To assure consumer‟s safety and high quality dairy products intended for export,
raw milk is to regularly analyze for the presence of antibiotic residues. Detecting
violative levels of antimicrobial residues in milk through the use of residue screening and
other qualitative tests can help prevent contaminated milk from entering the human food
chain. Although milk is not regulated at the individual cow basis, use of antibiotic
screening tests for individual cow milk is associated with reduced risk of bulk milk
residue incidence (McEwen et al., 1991).Different methods and assays for the detection
of residues of antimicrobials, mostly in cow milk, have been developed and validated,
whereas few studies have been carried out so far for detection of residues in sheep and
goat milk (Wang et al., 2006; Comunian et al., 2010).
To detect antibiotic residues, different kinds of methods were developed. These
methods can be classified in two main groups as of screening methods and
Padol et al., / Environ. We Int. J. Sci. Tech. 10 (2015) 7-28
19
chromatographic methods to detect as many antibiotics as possible at very low
concentration. The screening tests are generally performed by microbiological (Nouws et
al., 1999; Babapour et al., 2012), enzymatic and immunological methods (Strasser et al.,
2003). The working principle of screening methods is based on the variable susceptibility
of bacteria to different antibiotics. The antibiotic residue detection assays that are
currently available use different methods and test microorganisms (Mitchell et al., 1998).
Microbiological assays for the detection of antibiotic residues utilize bacteria such as
Bacillus stearothermophilus because of its high sensitivity to the majority of antibiotics.
The first test for establishing antimicrobial agent residues in milk (microbial inhibitor
test) was developed as early as 1952 (Mitchell et al., 1998). The developments of tests for
detection of antibiotic residues were initiated to establish the inhibitor agent levels in
milk, since the presence of these agents could cause the inhibition of the starter cultures
used in dairy industry (Navratilova, 2008). These methods are relatively cheap, simple to
carry out and capable of detecting a wide variety of antimicrobial agents. A drawback
which limits their use is a long incubation period. Therefore, rapid assays for antibiotic
agent detection in milk have been developed which enable obtaining the results in short
duration. The rapid tests are simple to perform, sensitive and specific. The rapid assay
developed include Penzyme test which was developed in 1980‟s. Later on, in 1988,
Charm II test for detecting 7 types of antimicrobial agents was introduced to the market,
followed by other rapid assays, e.g. the LacTec test (1991), SNAP test (1994), Beta Star
test, Charm Safe Level test (Mitchell et al., 1998) and Charm MRL-3 (Reybroeck et al.,
2011; Fejzic et. al., 2014).
Microbiological inhibition assays
In microbial growth inhibition test, standard culture of the test microorganism in a
liquid or solid medium is used; e.g. Geobacillus stearothermophilus var. calidolactis,
Bacillus subtilis, Bacillus megaterium, Sarcina lutea, Escherichia coli, Bacillus cereus or
Streptococcus thermophilus (Heeschen, 1993). Milk sample to be analysed is applied on
the agar surface and the plates are incubated for diffusion of the sample into the medium,
and if the sample contains inhibitor agents, inhibition of growth occurs of the tested
microorganism. The positive test is indicated either by formation of a clear zone of
inhibition around the disc or a change in the colour of medium (Reybroeck, 2014). At
present, many commercially produced microbial inhibitor tests are applied along with
selective rapid tests for milk sample screening in primary dairy industry for rapid and
precise detection of residues (Kozarova et al., 2009). The advantage of these methods is
that they have a wide detection spectrum, simple to carry out, reliability and they are
cheap and can be used for the screening of a large number of samples. Microbial inhibitor
tests detect a wider range of antimicrobial substances, including β-lactam antibiotics, and
give a result within 3 hours or less.
Receptor/ Protein binding assays
For detection of the β-lactam antibiotics residues in bulk or individual cow
milk samples, antibiotic specific receptor proteins or penicillin-binding proteins (PBP)
were successfully used in some methods and commercially available tests (Biacore
Padol et al., / Environ. We Int. J. Sci. Tech. 10 (2015) 7-28
20
analysis, Penzym test, Beta Star test, SNAP test, Charm Safe Level test and DELVO-X-
Press test and others) (Navratilova, 2008). Receptor binding assays are also a common
class of antibiotic residue screening tests. This type of assay involves a receptor protein
conjugated to an enzyme. The conjugate will bind to free β-lactam antibiotics that may be
present in the milk sample (Massova and Mobashery, 1998). One of the most common
receptor binding assays available commercially in market is the Beta star test. Beta Star
test is a receptor assay for rapid detection of the beta-lactam antibiotics penicillin,
ampicillin, amoxicillin, cloxacillin and cephapirin. This test is validated for use with raw,
commingled cow‟s milk (Movassagh and Karami, 2011).
Table.2. FSSAI recommended analytical methods for detection and quantitation of
antibiotic residues:
Sr. No. Antibacterials Analytical method
1 Beta-lactam antibiotics Bacillus stearothermophilus qualitative
disc Method-II
2 Chloramphenicol HPLC-MSMS method
3 Nitrofuran metabolites HPLC-MSMS method
4 Tetracyclines HPLC-UV/ MSMS method
5 Sulphonamides HPLC-MSMS method
6 Quinolones HPLC-MSMS method
7 Nitromidazoles HPLC-MSMS
Immunoassays
Immunoassays are quantitative or semi-quantitative methods characterized by
their high specificity, sensitivity, simplicity and cost-effectiveness, which make them
particularly useful in routine work. They are based on the specific reaction between
antibody and antigen. Nonisotopic immunoassays such as ELISA (Enzyme Linked
Immunosorbent Assay), FPIA (Fluorescence Polarisation Immunoassay), PCIA (Particle-
Concentration Immunoassay), PCFIA (Particle- Concentration Fluorescence
Immunoassay), and monoclonal-based immunoassays play an important role in
antibiotics screening immunoassay (Roeder and Roeder, 2000; Gaurav et. al., 2014).
These methods can also be applied for a preliminary identification of classes of
antibiotics (Sternesjo and Johnsson, 1998). Recently, Jiang and colleague (2013)
described a dual-colorimetric ELISA for the simultaneous detection of 13
fluoroquinolones and 22 sulfonamides with the detection limit of 2.4 and 5.8 ng/ml,
respectively. Further, ELISA is easy to perform, portable and reported to be very
sensitive and can be economical when many samples need to be analyzed.
Padol et al., / Environ. We Int. J. Sci. Tech. 10 (2015) 7-28
21
Monoclonal antibodies (Mab) also have been used to detect antibiotic residues in
milk. Dietrich and colleagues (1998) described an assay to detect ampicillin residues
using monoclonal antibodies raised against ampicillin-keyhole limpet hemocyanin
conjugate coupled by a glutaraldehyde in mice. Sensitivity and specificity of these Mabs
were tested with a direct competitive enzyme immunoassay, in which an ampicillin-
horseradish peroxidase conjugate prepared by a carbodiimide method served as the
labelled antigen.
Although the microbiological inhibition assay have been encourage because of
their simplicity, these methods lack specificity and allow for only semi-quantitative
measurements of residues detected and sometimes may produce false positives results
(Kurittu et al., 2000; Abbasi et al., 2011). Therefore, for quantitative measurements and
to detect the specific antibiotic and their metabolites, chromatographic techniques, such
as HPLC (high performance liquid chromatography), GC (gas chromatography) and
capillary electrophoresis (CE), have been developed to substitute microbiological assays
(Chen and Gu, 1995; Posyniak et al., 2005; Petkovska et al., 2006; Kantiani et al., 2009;
Kukusamude et al., 2010; Adetunji and Olaoye, 2012; Tona and Olusola, 2014). Also, to
avoid the condemnation of bulk milk quantity due to exceeding tolerance levels of
residues, it is required to analyze the sample with highly selective and sufficiently
sensitive analytical methods. The main drawbacks of chromatographic methods are: they
are expensive, non-portable, and therefore non-suitable to be use at farm level and require
expertise to operate. Hence, these methods are used only as confirmatory test and to
support the results of rapid screening tests. For the successful implementation of national
regulation and surveillance monitoring for antibiotic residues FSSAI recommend various
analytical and screening tests for detection of these residues in milk and other food
products (Table.2).
Possible strategies for prevention of antibacterial residues in Indian scenario
Preventing drug contamination of milk is the responsibility of every farm. Drug
residues can be avoided by a well planned drug use program. The sale of contaminated
milk will cause the responsible party to be subjected to severe penalties, including
suspension of permits and monetary loss. Milk with drugs can adulterate a whole
truckload or holding tank of milk.
1. Establishment of pharmacokinetics and withholding time for antibacterial used in
dairy animal to describe metabolism and distribution of drugs in different tissue
and milk. Withholding periods after treatment of cows with veterinary drugs
should be valued. The pharmacokinetics of a drug is also dependent on the vehicle
used in a certain drug formulation. Therefore the withholding time is valid for the
specific drug. Different withholding periods may be appropriate for two drugs
containing the same antibiotic.
2. One practical approach to cut down the residues in milk would involve good
hygiene and good management practices at farm and the milk processing units.
Modifying and implementing the good management practices is also very
Padol et al., / Environ. We Int. J. Sci. Tech. 10 (2015) 7-28
22
important for preventing the spread of disease among livestock which could
reduce the need of antibacterial use.
3. Evaluation and use of alternative to antibiotic growth promoter e.g. probiotic
microorganisms, immune modulators, organic acids (acidifers) and other feed
suppliments.
4. Although, Directorate General of Health Services under Ministry of Health and
Family Welfare has set out the policy for use of antimicrobials to combat
antimicrobial resistance in human and animal in India, there is no regulation or
policy regulating the use of antibacterials in animals for treatment or as growth
promoter. Establishing the use policy for antibacterial in animals will help for
monitoring and surveillance of the usage of these drugs.
5. Pharmacovigilance programme would be developed for veterinary
pharmaceuticals concerning the safety of veterinary medicines used for the
treatment, prevention or diagnosis of disease in animals.
6. Establishment of pharmacovigilance working group and an effective reporting
system involving veterinarians, immunologists, pharmacologists, toxicologists
and ecotoxicologists is an important prerequisite for the risk assessment of
antibacterial drug residues for human and environment.
7. Maintaining treatment records of cows in order to determine appropriate
withholding periods. This will also help to treat dry cow with long acting
substances so the withholding period can be adjusted if the dry period is shorter
than expected.
8. Recommendations of the drug manufacturer regarding dosage, route of
administration, treatment intervals and storage condition of antimicrobials should
be followed intimately because any deviation may contribute to extended
withholding periods.
9. Development and validation of rapid screening tests for detection of antimicrobial
residues in milk at individual cow basis to make sure that milk of individual cows
is free of inhibitors after the end of the withholding period.
Establishing regulatory standards and good management practices that reduce the
risk of antibiotic residues in milk supply are essential components of human food safety.
Within the last decade an increasing number of investigations covering antibiotic input,
occurrence, fate and effects have been published, but there is still a lack of regulations
and guidelines regarding use of antibiotics in veterinary practice in India. The issue of
antibiotic residues in food chain warrants the further policies and guidelines to address
the possible risk to public health and environment.
Authors' contributions: Amol R. Padol is responcible for colletion of review leterture, writing of
menuscript and also corresponding author; C. D. Malapure, Vijay D. Domple and Bhupesh P. Kamdi
are responsible for final editing of menuscript.
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